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1 AD Award Number: DAMD TITLE: Conditional Estrogen Receptor Knockout Mouse Model for Studying Mammary Tumorigenesis PRINCIPAL INVESTIGATOR: Sohaib A. Khan, Ph.D. CONTRACTING ORGANIZATION: University of Cincinnati Cincinnati, Ohio REPORT DATE: January 2002 TYPE OF REPORT: Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland DISTRIBUTION STATEMENT: Approved for Public Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation

2 Form Approved REPORT DOCUMENTATION PAGE OMB No Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA , and to the Office of Management and Budget, Paperwork Reduction Project ( ), Washington, DC AGENCY USE ONLY (Leave blank) 2. REPORT DATE 3. REPORT TYPE AND DATES COVERED January 2002 Annual (1 Jan Dec 01) 4. TITLE AND SUBTITLE 5. FUNDING NUMBERS Conditional Estrogen Receptor Knockout Mouse Model for DAMD Studying Mammary Tumorigenesis 6. AUTHOR(S) Sohaib A. Khan, Ph.D. 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION University of Cincinnati REPORT NUMBER Cincinnati, Ohio Sohalb.khan@uc.edu 9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSORING / MONITORING AGENCY REPORT NUMBER U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland SUPPLEMENTARY NOTES Report contains color 12a. DISTRIBUTION / AVAILABILITY STATEMENT 12b. DISTRIBUTION CODE Approved for Public Release; Distribution Unlimited 13. ABSTRACT (Maximum 200 Words) We have finished the construction of the ERa targeting vector and produced ES cell lines. The details of which are provided in the body of this progress report. The KO construct was electroporated into J-1 ES cells. 120 single clones were obtained after selecting the transfected cells with G418. The ES clone genomic DNA is being screened by PCR with ERKO 3' primers to detect the 5.2kb targeted ER-a allele. The targeted allele will be confirmed with ER/TK 3'-Neo primers, ERKO 5'-Neo primers and Southern blot with the 5' probe. The confirmed positive clone will be treated with Cre recombinase to delete the TK-Neo cassette. Following are the highlights of the progress report "* Establishment of breast cancer cell lines expressing different ERs. "* Identification of the target genes of estrogen and BPA in breast cancer cell line expressing only ERa. "* Contribution the F-domain of ERot in the estrogen-dependent recruitment of coactivators. "* Construction of the ERox targeting vector and generation of ES cell lines for generating conditional ERa KO mice. 14. SUBJECT TERMS 15. NUMBER OF PAGES Breast Cancer PRICE CODE 17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF ABSTRACT OF REPORT OF THIS PAGE OF ABSTRACT Unclassified Unclassified Unclassified Unlimited NSN Standard Form 298 (Rev. 2-89) Prescribed by ANSI Std. Z

3 Table of Contents Page Numbers 1. Front Cover 1 2. Standard Form 298, Report Documentation Page 2 3. Table of Contents 3 4. Introduction 4 5. Body Key research Accomplishments 8 7. Reportable Outcomes 8 8. Conclusions 8 INTRODUCTION 3

4 Breast cancer is among the most devastating diseases affecting women. The risk for a North American women getting breast cancer has doubled since 1940, and at present one woman in eight is at risk of developing the disease. It is generally believed that estrogens play a significant role in the development of breast cancer. The mitogenic action of estrogen is amplified by the hormone-dependent transcription factor, estrogen receptor (ER). Because of its pivotal role in the normal physiology of breast, ER has become a target for pharmacological intervention in breast cancer. This contention is strengthened by observations that the estrogen receptor is a hormone dependent transcription factor that regulates the expression of growth factors and protooncogenes in breast tumor cell lines. Moreover, the growth and progression of many breast cancers are dependent upon estrogen, making measurement of ER-oa standard in the treatment decisions for patients with breast cancer. The ER-positive breast tumors are generally associated with faster growing and more aggressive tumors than the ER-positive tumors, which can be controlled with antiestrogen therapy. Towards understanding the role of ER in breast tumorigenesis, we have initiated a program, using transgenic mouse technology to ablate ER-oa gene in the mammary gland. This mouse model is expected to be of great use in addressing the role of estrogen receptor in mammary tumorigenesis. In the previous Progress Report we described our work with ERoa mutant (K303R); yeast two-hybrid studies with ERa /ERfP heterodimerization and nongenomic action of estradiol. In the present report, we will only present the progress summary. The current Progress Report will focus on our accomplishments related to the main focus of the DOD-funded project, i.e. generation of conditional ERoa knock out (KO) mice. BODY 1. Study on human ER-K303R mutant: We have used the cdna encoding ER (K303R) to stably transfect an ER null cell line called C4-12, using the G418 selection system. The stable cell line was tested for expression of the transgene by Western blot analysis. 2 Protein-protein interactions between ERa and ERP. We have expanded the yeast two-hybrid experiments to study the interaction between ERs and the receptor coactivators and concluded that the F-domain of the receptor affects the estrogen-dependent interactions between p160 coactivators and the estrogen - bound ERs. 3. Studies with C4-12 breast cancer cells. In this study, our most exciting achievement has been the establishment of breast cancer cell lines expressing ERoa, ERP3, K303R or ERoL3f. We have now systematically begun to analyze these cell lines to determine the target genes for ERs. In the first set of such an experiments, we challenged the ERoa expressing cell line with vehicle, estradiol or an environmental estrogen bis phenol-a (BPA) 4

5 and analyzed the gene expression pattern with microarray technology. As presented in Table-I, an interesting expression pattern of the target genes emerged. The intriguing observation is that some genes that are induced by estradiol are actually inhibited by BPA and the two ligands showed a different expression patterns. We have further confirmed the expression of some of these genes by real time PCR and focused on one of the protooncogenes, c-myb whose expression has been previously seen in breast cancer patients. We have cloned and sequenced its promoter, which revealed no consensus estrogen response element (ERE). It is likely that c-myb is induced by estradiol and BPA through AP-1 promoter elements. 5. Estrogen receptor-(x gene-targeting vector and ES cell lines: We have finished the construction of the ERox targeting vector and produced ES cell lines. The details are provided below: Estrogen Receptor-a -gene targeting vector: Construction & characterization. Spel Exon3 HpaI W ild-typ e == ER-a Locus I BamHl PmIl NheI Eco47III BamHI BamHl construct T Spel iexon3 HpaI Clal ClaI BamHl Pmll NheI Eco47III BamHI HSV-TK gene Floxed TK-Neo Figure 1 Wild-type ER-a exon3 locus and targeting construct In order to make the targeting vector, we originally focused to delete exon 2 and obtained a mouse genomic DNA clone harboring a 10kb BamHI fragment of mouse ERa. We however revisited the issue and decided to focus on deleting exon 3. This was prompted by the fact that knockout mice carrying exon 2 deletion in the genomic KO (reported by Ken Korach) some low levels of truncated ERa expression persisted. We obtained the exon 3 DNA fragment (pbc-era-bamhl) from Jan Ake Gustafsson (Karolinska Institute). The pbc-era-bamhl construct was amplified 5

6 and analyzed by restriction enzyme digestion and sequencing (fig. 1). We have confirmed that the 9.2 kb insert is the ERa genomic fragment and harbors exon3 (fig. 2) kb ER-a 4-9kb genomic DNA 4-7kb 4-6kb 4-5kb 3.4kb pbc-sk -* vector 4-4kb 4-3kb Figure 2 pbc-era 9.2kb plasmid digestion. Lane 1: BamHI and SpeI digestion. Lane 2: BamHI digestion. Lane 3: BamHI and NotI digestion. Lane 4 1kb marker (Promega). Utilizing a series of cloning steps, we designed the ERa conditional Knockouttargeting construct according to a scheme presented in Figurel. In order to screen the positive ES cell clones more efficiently, we introduced a negative selection gene, HSV TK, into the Cla-I site of pbcsk vector (Stratagene). The "Floxed TK-Neo cassette" was inserted into the Eco43111 site on the 3' side of exon3. The 46bp LoxP-BamHI fragment was inserted into the Nhel site on the 5' side of exon3. The targeting construct was analyzed for its accuracy by restriction enzyme analysis and DNA sequencing (fig 3). The complete sequence of the 12.6 kb "targeting construct' was sequenced from 5' and 3' ends of the genomic DNA insert. We then compared this DNA sequence with the Celera gene bank using the BLAST software and found 97% sequence homology with the mouse genome. At this stage we decided to electroporate the targeting construct into the ES cells Electroporation of the tar-geting construct into ES cells. The KO construct was first linearized with Pmll and electroporated into J-1 ES cells. 120 single clones were obtained after selecting the transfected cells with G418 at a concentration of 240ptg/ml. The ES clones were cultured in 24-well plate to full confluency and lysed overnight for the isolation of their genomic DNA. PCR primers were designed to detect the entire targeting construct (fig. 4). The ES clone genomic DNA is being screened by PCR with ERKO 3' primers to detect the 5.2kb targeted allele. The targeted allele will be confirmed with ER/TK 3'-Neo primers, ERKO 5'-Neo primers and Southern blot with the 5' probe. The 6

7 confirmed positive clone will be treated with Cre recombinase to delete the TK- Neo cassette bp A 4000bp bp bp bp bp bp " 3000bp - A B Figure 3 pbc-erctko construct BamHI digestion. Two BamHI sites have been introduced into the KO construct at LoxP-BamHI site and the 3' end of Floxed TK-Neo cassette. 4 bands are generated by BamHI (1.9kb, 3.9kb, 4.7kb and 5.4kb). Panel B is a longer run of Panel A. Lane 1 is Promega lkb marker. Lane 2 shows KO construct BamHI digestion. 5' probe BamHI BamHI Floxed TK-Neo BamHI ExonB3 BaMHI PmII 4 5.2kb kb l--- 4kb -4 ERKO 5'-Neo primers -4 ERKO 3'-Neo primers -0 ERKO 3' primers Figure 4 Strategies to screen and confirm the targeted allele. The ES clones will be Screened with ERKO 3' primers for 5.2kb targeted allele. The targeted allele will be confirmed with ERKO 3'-Neo primers, ERKO 5'-Neo primers and Southern blot with 5' probe. Proposed Changes for the Future Work: 7

8 We are not proposing any changes in the experimental plan. After confirming the integration of the targeting construct into the ES cells, they will be used to generate the mice carrying Floxed ERox gene. Once the colony is established, they will be crossed with transgenic mice carrying Cre recombinase gene under the why acidic protein (WAP) promoter to obtain ERax conditional KO mice. Key Research Accomplishments: * Establishment of breast cancer cell lines expressing different ERs. * Identification of the target genes of estrogen and BPA in breast cancer cell line expressing only ERoa. * Contribution the F-domain of ERax in the estrogen-dependent recruitment of coactivators. * Construction of the ERoa targeting vector and generation of ES cell lines for generating conditional ERoa KO mice. REPORTABLE OUTCOMES Singleton, D., and Khan, S. "Estrogen receptor dependence of MAPkinase activation in breast and uterine tumor cells Annual Meeting of the "ENDOCRINE SOCIETY', Denver, Co (2001) Singleton DW, Feng Y, Burd C, Khan SA. Activation of Ras/ERK1/2 by Estrogen Receptor Ligands is Necessary But Not Sufficient to Promote DNA Synthesis in a Human Endometrial cell line. (Manuscript submitted to Endocrinology) Singleton DW and Khan SA. Xenoestrogen Exposure and Mechanisms of Endocrine Disruption. (Manuscript submitted to Frontiers in Bioscience Singleton DW, Feng Y, Burd C, Khan SA. Nongenomic Action of Estrogen Receptor Ligands Is not Sufficient to Induce Proliferation of Human Endometrial Adenocarcinoma Cells. (Abstract submitted for presentation at The Endocrine Society Meeting, June 2002) CONCLUSIONS Estrogen receptor plays a key role in breast cancer. However, little is known about the involvement of the receptor in the progression of breast tumorigenesis. Towards achieving our overall goal of generating ER knockout mice for studying mammary tumorigenesis, we had proposed to generate ER-oa conditional knockout mice. We have successfully generated the targeting construct for use in generating ERax knockout mice. We have also worked on generating ER-specific breast cancer cell lines towards understanding the role of different ER dimmers in estrogen signaling. Using microarray analysis we have identified specific genes for ERoa. Finally, we have used the yeast genetic system to explore the role of the F-domain of ER in the recruitment of coactivators for the control of estrogen gene expression. 8